Assay

ABSTRACT

A process for identification of type-specific polynucleotide sequences in a sample, the process comprising the steps of (1) contacting polynucleotides from the sample, or derived from the sample, with a plurality of type-specific probes in the context of a solid support, and detection of any type-specific hybridisation; and (2) contacting polynucleotides in the sample, or derived from the sample, with type-specific primers for those types capable of being detected by the hybridisation step in step 1, but not so detected, in a type-specific amplification reaction.

The present invention relates to a method for nucleic acid detection andanalysis.

In particular the invention relates to methods for the detection andtyping of nucleic acid, e.g. for diagnosis, and identification ofmutations in genes associated with disease.

The importance of accurate and comprehensive genotyping analysis isexemplified by the typing of human papillomavirus (HPV).Papillomaviruses are small DNA tumour viruses, which are highly speciesspecific. So far, over 100 individual human papillomavirus (HPV)genotypes have been described. HPVs generally infect either the skin(e.g. HPV-1 and -2) or mucosal surfaces (e.g. HPV-6 and HPV-11) andusually cause benign tumours (warts) that persist for several months oryears. Such benign tumours may be distressing for the individualsconcerned but tend not to be life threatening, with a few exceptions.

Some HPVs are, however, associated with more serious disease such ascancers. The strongest positive association between an HPV genotype andhuman cancer exists between HPV-16 and HPV-18 and cervical carcinoma.Individuals with, for example, HPV 16 and/or 18 infection are consideredto be at high risk of developing the disease. Cervical cancer is themost common malignancy in developing countries, with about 500,000 newcases occurring in the world each year.

At present large scale nucleic acid typing is generally carried out byprobe array hybridisation.

By way of example, in the HPV field, Sahli recently disclosed a methodfor diagnosis of HPV infection by PCR amplification of HPV DNA followedby probe array hybridisation and DNA sequencing [R. Sahli, WHO meetingHeidelberg, Germany, 6-7 March 2001. “Assessment and Harmonisation ofLaboratory Diagnostic procedures related to HPV Vaccine research andDevelopment”]. The method comprises a first PCR step to amplify HPV DNAwithin a sample using broad spectrum primers specific to HPV in general,but not specific to any HPV type in particular. This is followed byreverse blot hybridisation of the PCR fragments so generated withmultiple type-specific oligonucleotide probes bound to a solid support.The hybridisation of a PCR fragment with a type-specific oligonucleotideallows specific HPV types in the sample to be detected. The use ofhybridisation techniques in this way allows the screening of a sampleagainst multiple HPV probes after a single PCR, and avoids the need formultiple individual specific PCR reactions.

For those samples identified as HPV-positive by the initial PCRreaction, but for which there is no positive hybridisation reaction withan HPV specific probe, direct sequencing of the DNA is carried out toidentify the HPV type, or confirm the presence of a new type. Sahlidiscloses that HPV-positive but hybridisation-negative amplicons alwayscorresponded to HPV types or subtypes not represented in thehybridisation specific array, as determined by sequencing.

Screens involving a similar hybridisation detection systems are knownfor identification and typing of a number of bacterial and viral genes,such as those derived from HIV-1, HCV, HBV, Helicobacter pylori andMycobacteria, mycoplasma, along with typing of genes such as p53,involved in cancer.

The present invention provides an improved method for nucleic aciddetection and typing.

In a first aspect, the invention relates to a process for identificationof type-specific polynucleotide sequences in a sarnple, the processcomprising the steps of:

-   -   (i) contacting polynucleotides from the sample, or derived from        the sample, with a plurality of type-specific probes in the        context of a solid support, and detection of any type-specific        hybridisation; and    -   (ii) contacting polynucleotides in the sample, or derived from        the sample, with type-specific primers for those types capable        of being detected by the hybridisation step in step 1, but not        so detected, in a type-specific amplification reaction.

In a preferred embodiment the invention relates to a process foridentification of type-specific polynucleotide sequences in a samplecomprising the steps of

-   -   (i) amplification of nucleic acid from the sample using broad        spectrum primers;    -   (ii) contacting polynucleotides obtained from (1) with a        plurality of type-specific probes in the context of a solid        support, and detection of any type-specific hybridisation; and    -   (iii) contacting polynucleotides in the sample, or derived from        the sample, with type-specific primers for those types not        detected by the hybridisation step in step 2, in a type-specific        amplification reaction.

Preferably the process additionally comprises a further step 1b, whereinnucleic acid amplified by broad spectrum primers in step 1 is analysedto confirm the presence of general polynucleotide types of interestprior to hybridisation. Only those samples positive for the generalpolynucleotide types of interest are screened in step 2. Preferably theamplimers obtained from (1) are contacted with a mixture of generalprobes which are capable of recognising a broad range of types,preferably in a microtitre plate format as outlined in Kleter et al [Am.J. Pathology (1998), 153: 1731-1739].

Optionally the process comprises a detection signal amplification stepwhich is not type-specific.

The invention particularly relates to a process for identification ofviral types in a sample, more particularly an HPV type.

The invention also relates to a method for identification of individualsat risk from disease, comprising typing analysis of a sample from theindividual using the method of the invention.

The invention further relates to diagnostic kits comprising at least oneset of suitable broad spectrum primers in combination with at least oneset of type-specific primers, optionally in combination with meansappropriate to carry out a type-specific hybridisation reaction.

We have determined that current analysis processes provide only anincomplete picture of the disease risk associated with a given sample.Certain samples may contain for example, viral types with a highassociation with disease which are not detected at the hybridisationstep, even though the hybridisation step contains probes specific forsuch type detection.

In the case of HPV, for example, we have determined that samples whichappear by hybridisation analysis only to contain a type having only alow association with disease also comprise high risk types, despite thepresence of specific probes designed to those high risk types. Currenttesting routines would not identify such high risk types, and such typesmight not be identified in several samples.

Accordingly the present invention is preferably concerned with typingsamples which comprise or may comprise multiple polynucleotide types.

It has been determined that, unless a type of interest has beenpositively identified by hybridisation, it is not possible to assumethat such a type is absent from the sample. This is true even when aspecific probe for the type of interest is used during the hybridisationphase. As such, in the present invention, a selective, type-specificamplification step is introduced after any typing process involving ahybridisation step for the detection of specific high risk types notidentified by the hybridisation screen. The use of such a specificamplification step allows a more complete determination of the typespresent, for example a more complete determination of HPV infection,than was previously available.

As for hybridisation-based detection systems, direct sequencing methodssuffer from a similar drawback; where there is multiple infection thelow level concentration of a type may not be detectable. Accordingly,sequencing alone cannot provide a definitive answer to types present ina sample, due to limited sensitivity. A further type-specificamplification stage is also required.

Without wishing to be constrained by theory, it is thought thatcompetition between polynucleotide types in a sample can lead to certainpolynucleotide types being undetected. Competition might occur at anumber of different stages in the process. By way of example, in thecase of HPV typing using broad spectrum PCR primers, if one HPV type ispresent in great molar excess over another type it is likely that theminor type will be outcompeted and could remain below the detectionlimit of the assay. Additionally, since different HPV genotypes maycontain slightly different nucleotide sequences at the primer targetregions, each genotype will be preferentially amplified by a subset ofPCR primers from the available broad-spectrum primer pool. Furthermore,at the signal detection level, the detection of hybridisation between atype-specific probe and its corresponding type-specific polynucleotidetarget may also be affected where there are multiple types in a sample.

Suitably the present invention comprises a hybridisation/detection step,the result of which is sensitive to competition between polynucleotidetypes in a sample, for example competition for detection probes oramplification primers.

Suitably the hybridisation/detection step of the present invention iscapable of giving a false negative result when used to analyse a samplecomprising mixed polynucleotide types.

A type-specific polynucleotide sequence of the present inventionrepresents a specific subset of a broader class of related sequences. Inparticular a given type is preferably characterised on the basis ofsequence and/or hybridisation characteristics, and may be distinguishedfrom members of a broader class on the basis of these parameters.

In the cases of viruses a ‘type’ is preferably a genotype, andidentification of a type-specific polynucleotide sequence in a sample issuitably genotype identification. For example, in the case of HPV, viralisolates that display a sequence difference of more than 10% to anypreviously known type in the L1 gene are classified as differentgenotypes (Chan et al, Journal of Virology (1995) 69:3074-3083).However, isolates may be further classified, and HPV isolates thatdiffer between 2 and 10% are classified as subtypes, while if thevariation is below 2% the isolates are classified as variants. As such,any reference to typing herein includes reference to analysis of types,subtypes and variants, as appropriate.

In the case of single gene mutations, for example, a type may be aspecific mutation in a gene associated with disease. In the case of theclass of the p53 gene, different mutations in the gene are different p53types.

As such, a type-specific polynucleotide sequence may be a specific genesequence or one of a group of closely related sequences such as a type,subtype or variant.

Preferably, the method of the invention is used for the typing of HIV-1,hepatitis C virus (HCV), hepatitis B virus (HBV), cytomegalovirus (CMV),Epstein-Barr virus (EBV), hepatitis D virus (HDV), hepatitis G virus(HGV), Herpes simplex virus (HSV), Human herpes virus (HHV),Varicella-zoster virus (VZV), Helicobacter pylori, Mycobacteria,mycoplasma, rotavirus, and typing of genes associated with disease suchas p53. Other preferred typing targets include viruses and genes forwhich different types correlate with disease development and/orseverity. Most preferably the present invention is used in typing ofHPV. However, it will be appreciated that the present invention is nottechnically limited to the source of the nucleic acid, and may be usedto type nucleic acid from any gene or virus, as appropriate.

The method of the invention uses a sample, which is suitably biologicalmaterial, such as a tissue sample, taken from an individual being testedfor infection and/or risk of disease. Body fluids such as blood andurine may also be used in the process of the invention. There isgenerally no requirement to take a sample by any invasive surgicalprocess for DNA typing, and swabs may be used to obtain such samples,for example. Non invasive, non surgical methods for obtaining samplesare preferred. Other suitable methods for obtaining samples fromindividuals for typing are well known in the art.

In the process of the invention sample polynucleotide/nucleic acid andprobe are contacted to allow specific hybridisation, if any, to takeplace.

The polynucleotide for analysis may be used directly from the sample or,more preferably, after a polynucleotide amplification step (eg PCR)step. In some cases it may be necessary to transcribe RNA to DNA beforeamplification. In both latter cases the amplified polynucleotide isderived from the sample.

Hybridisation of the polynucleotides may be carried out using anysuitable hybridisation method and detection system. Examples ofhybridisation systems include conventional dot blot and Southern blots,for example. Preferred is a reverse hybridisation approach, whereintype-specific probes are immobilised on a solid support, and amplifiedpolynucleic acids are labelled in order to detect hybrid formation. Mostpreferred is the LiPA system described in WO 99/14377, and in Kleter etal, [Journal of Clinical Microbiology (1999), 37(8):2508-2517], thewhole contents of which are herein incorporated by reference. In thissystem the oligonucleotide probes are immobilised on a solid support inparallel lines. However, other reverse hybridisation systems may also beemployed, for example, as illustrated in Gravitt et al, [Journal ofClinical Microbiology (1998)36(10):3020-3027] the contents of which arealso incorporated by reference.

Preferably the polynucleotide sample is screened simultaneously againstmultiple probes, under the same conditions of hybridisation and washing.

The type-specific probe of the present invention is suitably a singlestranded oligonucleotide designed to hybridise to nucleic acid from agiven type, such as a viral genotype, which enables identification ofthat type in a sample.

Type-specific probe sequences are well known in the art, and any suchsuitable sequences can be used in the present invention. In the case ofHPV, for example, suitable type-specific probes are disclosed inWO9914377. The invention is not restricted to the nature or origin ofthe type-specific probes that are used in hybridisation step in thepresent invention.

Type-specific probes may be attached to any suitable solid support, suchas microtitre dishes, membranes such as nylon or nitrocellulose,microspheres or chips. Other suitable supports are well known in theart. The type-specific probes may be modified in order to allow fixationor improve hybridisation efficiency. Such probes are thus be used in thecontext of a solid support to contact polynucleotides of interest in theprocess of the invention.

There is no restriction on the detection system that may be used todetect a type-specific hybridisation reaction. Either the probe ornucleic acid may be labelled. Preferably the nucleic acid to be screenedis labelled. Suitable detection systems include radioactive detectionby, for example ³²P and ³⁵S, or non-isotopic detection systems whichuse, e.g. fluorescence. A suitable non-radioactive detection method isdisclosed in EP-A-667918.

The presence of a nucleic type in a sample is confirmed by a positivehybridisation reaction. However, the present invention demonstrates thatthe absence of a positive result cannot be taken to indicate the absenceof a viral type. Accordingly, the process of the invention provides atype-specific amplification step using type-specific primers followingthe hybridisation screening step.

A primer is suitably a single stranded oligonucleotide sequence whichserves to act as a startpoint for the initiation of a primer extensionproduct which is complementary (either 100% or partially) to the nucleicacid strand to be copied. Suitable primers for the amplification ofspecific DNA types may be designed using methods standard in the art. Inaddition, type-specific primers for a number of viral and gene types arewell documented.

Preferred type-specific primers for amplification of HPV types aredescribed in Baay et al., [Journal of Clinical Microbiology, March(1996), 745-747], Karlsen et al [Journal of Clinical MicrobiologySeptember 1996, vol 34, no 9, 2095-2100] and Yoshinouchi et al [Journalof Clinical Microbiology. November 1999, Vol37, N°11, p 3514-3517.], thesequences of which are incorporated by reference herein. Preferred areany HPV primers specific for genotypes associated with high risk ofcervical cancer, such as, but not limited to, HPV 16, 18, 31,33, 45, 52,58, 35, 56, and 59.

Type-specific amplification is suitably carried out by the PCR process.The PCR process is well known and documented in the art. Theamplification comprises repeated cycles of heat denaturation, annealingof primers to sequences that flank the DNA sequence to be amplified, andextension of the annealed primers with DNA polymerase. The primershybridise to opposite strands of the target sequence and are orientedsuch that DNA synthesis by the DNA polymerase proceeds across the regionbetween the primers. Amplification of DNA by the PCR reaction isdisclosed in U.S. Pat. Nos. 4,683,202 and 4,683,195, the contents ofwhich are incorporated by reference. In addition, techniques for theanalysis of PCR products are standard in the art, such as, for example,sequence analysis or restriction analysis.

Nucleic acid amplification is, however, not limited to PCR and may alsobe carried out by other suitable methods, such as NASBA (Compton,J(1991) Nature 350:91-92) and LCR (Backman K et al (1989) EP-A 0320 308).Other suitable amplification methods are well known in the art.

Type-specific amplification is suitably followed by a detection step toconfirm the presence or absence of an amplimer.

Preferably the type-specific amplification of the present invention is aquantitative process. Quantitative PCR, for example, allows the level ofa given nucleic acid type, such as a viral genotype, to be determinedand to be correlated with the likelihood of disease and/or diseaseprevention. In particular, this is important diagnostically when diseaserisk is increased above a certain threshold of viral load, for example.The correlation between viral load and disease progression, along withquantitative PCR techniques is illustrated in Swan et al. [Journal ofClinical Microbiology, April 1999,37(4):, 1030-1034] and Josefsson etal. The Lancet, June 2000. 355:2189-2193], incorporated herein byreference in respect of such techniques.

The nucleic acid for type-specific amplification is preferably obtaineddirectly from the original sample. Amplification may also be carried outon nucleic acid derived from the sample. For example, where the originalsample is RNA, then amplification may be carried out after reversetranscription of the RNA to cDNA. Alternatively, the amplification maybe carried out on nucleic acid amplified from the original sample by abroad primer set, for example.

Preferably there is amplification of sample nucleic acid prior tohybridisation screening. Such amplification may be through PCR, orrelated methods, as discussed above.

Preferably broad spectrum primers are used in any pre-hybridisation stepto amplify multiple nucleic acid members of a class of interest, such asHPV DNA. Broad spectrum pruners are thus any primers, or groups ofprimers, which allow amplification of multiple types of nucleic acidfrom class of related sequences. Broad spectrum primers thus encompassprimers which amplify types within a species, subtypes within a type andvariants within a subtypes, for example.

In the preferred case of HPV analysis, preferred broad spectrum primersallow for amplification of DNA from at least 30 HPV types, preferably 40types, more preferably 50 types or even more. Preferably the broadspectrum primers allow for amplification of polynucleotides fromdifferent genotypes. Examples of suitable primers are given in Kleter etal [American Journal of Pathology (1998)153(6):1731-1738], andreferences comprised within, the whole contents and primer sequences ofall of which are incorporated by reference. In particular, preferred arethe SPF1 and SPF2 primer sets as described in Kleter et al (supra).Other methods and primers for broad spectrum amplification of HPV DNAare given in WO 99/14377, the whole contents of which are incorporatedby reference.

The present invention also relates to a process in which there isamplification of nucleic acid pre-hybridisation and/or signalamplification post hybridisation.

The present invention also relates to kits suitable for use in thetyping process described above. Kits of the present invention suitablycomprise components for viral or gene typing, preferably HPV typing.Preferably kits comprise broad spectrum primers and at least one set ofprimers which are type-specific. More preferably kits comprise HPV 16and/or HPV 18 Type-specific PCR primers. Preferably kits also comprise asolid support to which are attached HPV Type-specific probes. Kits mayalso comprise any necessary hybridisation and wash and detectionsolutions.

For the avoidance of doubt all publications, including but not limitedto patents and patent applications, cited in this specification areherein incorporated by reference as if each individual publication werespecifically and individually indicated to be incorporated by referenceherein as though fully set forth.

The present invention is now illustrated with respect to the followingnon-limiting example;

EXAMPLES 1 Detection of HPV Genotypes

Patients

Eleven patients were selected from a larger group, based on the presenceof multiple HPV genotypes comprising HPV16, 18, 31, 33, 35, 51, 52, or58, as determined by SPF₁₀ PCR and LiPA [Quint WGV et al., Journal ofPathology (2001), 194:154-158]. The selection of samples was based onthe detection of those genotypes, for which reliable type-specific PCRprimers were available [Baay MF et al. Journal of Clinical Microbiology(1996), 34:745-747; Walboomers JM et al, Journal of Pathology(1999),189:12-19]. The specificity of each of these type-specific PCRs wasreconfirmed by analysis on recombinant plasmids, containing completegenomic DNA from 13 different genotypes, including HPV 16, 18, 31, 33,35, 39, 51, 52, 56, 59, 58, 59, 66, and 68.

DNA Isolation, PCR and Genotyping

From each patient, a cervical biopsy specimen was obtained. DNA wasisolated by guanidinium isothiocyanate treatment and capture onto silicaparticles [Boom R et al, Journal of Clinical Microbiology (1990),28:495-503]. HPV DNA was amplified by SPF₁₀ PCR and amplimers wereanalysed on the HPV-LiPA as described [Kleter B et al Journal ofClinical Microbiology (1999), 37:2508-2517]. Subsequently, the same DNAisolate was tested by type-specific PCR primer sets of the types thatwere found positive by LiPA.

Results

The results of the SPF10-LiPA and the type-specific PCRs in biopsyspecimens are summarized in Table 1.

In patient 9, HPV 16 and 31 were detected by type-specific PCR in thebiopsy specimen, while these types were not identified by SPF₁₀-LiPA.Similarly, in patient 11, SPF₁₀-LiPA identified only HPV 16 in thebiopsy specimen, while type-specific PCR detected additional types 51and 52. TABLE 1 Identification of HPV genotypes by SPF₁₀-LiPA andtype-specific PCR primers. Biopsy Types found by Type-specific SampleSPF₁₀-LiPA PCR 1 16, 33 16, 33 2 18, 31 18 3 18 18 4 16, 31 16, 31 5 16,58 16, 58 6 16, 18 16, 18 7 16, 31, 52 16, 31, 52 8 16, 31 16, 31 9 1818, 16, 31 10 18 18 11 16 16, 51, 52

1. A process for identification of type-specific polynucleotidesequences in a sample, the process comprising the steps of: (i)contacting polynucleotides from the sample, or derived from the sample,with a plurality of type-specific probes in the context of a solidsupport, and detection of any type-specific hybridisation; and (ii)contacting polynucleotides in the sample, or derived from the sample,with type-specific primers for those types capable of being detected bythe hybridisation step in step 1, but not so detected, in atype-specific amplification reaction.
 2. A process according to claim 1additionally comprising a first step of amplifying polynucleotides fromthe sample using broad spectrum primers and subsequent use of saidamplified polynucleotides in step (i)
 3. A process according to claim 2,wherein polynucleotide sequences amplified by broad spectrum primers areanalysed to confirm the presence of general polynucleotide types ofinterest prior to hybridisation.
 4. A process according to claim 1wherein the type specific amplification is quantitative.
 5. A processaccording to claim 1 wherein the type specific primers are specific forone of HPV 16, 18, 31,33, 45, 52, 58, 35, 56, and
 59. 6. A method foridentification of an individual at risk from disease, the methodcomprising detecting and typing of a polynucleotide correlated with thedisease in a sample from the individual using the process according toclaim
 1. 7. A diagnostic kit for use in the process of claim 1comprising at least one set of suitable broad spectrum primers incombination with at least one set of type-specific primers.
 8. A kitaccording to claim 7 comprising HPV 16 and/or HPV 18 type-specific PCRprimers.
 9. A process, method or kit according to claim 1 for the typingof HPV, HIV-1, HCV, HBV, CMV, EBV, HDV, HGV, HSV, HHV, VZV,Helicobacterpylori, Mycobacteria, mycoplasma, rotavirus or gene p53. 10.A process, method or kit according to claim 9 for the typing of HPV.